Technical Field
The present invention is generally related to the field of designing circular permuted proteins and is more specifically related to the field of designing circular permuted proteins using two engineered Mu transposons for easy construction of random circular permuted proteins.
Prior Art
Circular permutation is an important method used in protein engineering that produces protein variants whose backbone is linked, via the N and C termini, and cleaved elsewhere to introduce new termini (1, 2). For example, circular permutation of thermosome and β-lactamase (BLA) created protein variants with higher expression levels, due to increased resistance to proteolysis (3, 4). Circular permutation can also be used to alter substrate specificity, as was the case with lipase B from Candida antarctica (CALB) (5). A circular permuted guest protein inserted into a host protein increased allosteric interactions between these two domains by altering their relative orientations (6, 7). These studies delineated the importance of circular permutation as an alternative to other protein engineering tools, such as random mutagenesis (8, 9), DNA shuffling (10), and site-directed mutagenesis (11) for creating variants with desired functions. When constructing circular permuted proteins, a peptide linker is usually incorporated at the genetic level (12). Fortunately, about 50% of single domain proteins have their N- and C-termini proximal (13), suggesting that circular permutation can be applied to a broad range of proteins. And it is possible that a backbone linkage between the two termini further apart from each other can still be made with a relatively long linker, increasing the applicability of circular permutation. While one can produce circular permutants chemically (2), recombinant circular permutation is usually more straightforward.
Not all circular permutations lead to correctly folded variants (14). In fact, the success of circular permutation heavily relies on the locations of new termini. Usually, loops distant from active sites, or other functionally sensitive regions of the protein, are preferred (7, 15). Backbone flexibility of parental proteins, which is well represented by B-factors, can also serve as an important guideline when selecting new termini for the circular permutant (3). Rational selection requires significant structural knowledge, which is not always available, and there is no well-established strategy to ensure the success of a rationally designed circular permuted protein. Instead, thorough examination of locations of potential new termini along the polypeptide backbone, followed by construction and subsequent evaluation of the random circular permuted variant, is often necessary.
A more powerful approach to designing circular permuted proteins is to use combinatorial methods to generate libraries that survey a much larger number of potential candidates. Combinatorial construction of a random circular permuted library typically involves a few characteristic steps: The 5′ and 3′ ends of a target gene is genetically attached with oligonucleotide sequences containing the same restriction enzyme site. Subsequently, digestion of the restriction enzyme site using an appropriate enzyme creates terminal sticky ends, which are used for DNA circularization of the target gene. The terminal nucleotide modification also introduces sequences encoding a backbone peptide linker upon DNA circularization. This is followed by treatment of a non-specific endonuclease optimized to introduce a single cut into the circular DNA construct. The result is linear permuted genes containing gaps and nicks that are later repaired by ligases and polymerases, creating blunt ended DNA (16). The resulting DNA construct is subsequently blunt-end ligated with a plasmid, as shown in FIG. 2. FIG. 2 is a schematic of circular permutation using restriction enzyme. Linear DNA with sticky ends is first cyclized, followed by treatment with restriction enzyme to create randomly cut linear DNA with nicks and gaps. Finally treatment with T4 DNA ligase and T4 DNA polymerase to repair nicks and gaps and create blunt ended circular permuted DNA. Though widely used, even this design improvement compared to rational design, has limitations—for example, optimizing conditions to introduce single cuts is technically difficult (17). In addition, random DNA cuts using a non-specific endonuclease such as DNase are sometimes difficult to control, and frequently result in truncations and duplications of parts of the DNA sequence (18-20). Furthermore, the blunt ended DNA that is generated after treatment with ligase is not preferred for ligation, as it may facilitate self ligation.
Alternatively, to mitigate the occurrence of truncations and duplications that occur when a non-specific endonuclease is used, one can employ a transposon for better control (21). While this method alleviates truncation and duplication, an unwanted 18 amino acids, resulting from DNA encoding the recognition binding sequences on the transposon, remained. Though proteins can sometimes tolerate additional amino acids appended to their C termini, these appendages usually serve a purpose—for example, purification. However, the additional sequence adds no value to the circularly permuted protein. The wide ranging use of transposons have been exhaustively reported elsewhere (21-25), and will not be elaborated on in this disclosure. However, recently, the present inventors also engineered a new transposon called MuST that facilitates facile construction of a random domain insertion library. The MuST transposon was designed with specific restriction sites at the 5′ and 3′ ends of the transposon, between the cleavage recognition sequence and the recognition binding site of the transposon, which allows for optimal control of composition and length of inter-domain linker residues (26). FIG. 11 shows wild type Mu Transposon with R1 and L1 recognition binding sites sequence. Blue arrows indicate Mu Transposon cleavage sites.
Accordingly, there is a need for new and different methods for designing circular permuted proteins. It is to this need and others that the present invention is directed.